Professional Documents
Culture Documents
* Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety
information.
†
Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a
working solution.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of
RNeasy Micro Kit is tested against predetermined specifications to ensure consistent
product quality.
Technical Assistance
At QIAGEN, we pride ourselves on the quality and availability of our technical support.
Our Technical Service Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in sample and assay technologies and the use of
QIAGEN products. If you have any questions or experience any difficulties regarding
the RNeasy Micro Kit or QIAGEN products in general, please do not hesitate to contact
us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as well as
to the researchers at QIAGEN. We therefore encourage you to contact us if you have
any suggestions about product performance or new applications and techniques.
For technical assistance and more information, please see our Technical Support Center
at www.qiagen.com/goto/TechSupportCenter or call one of the QIAGEN Technical
Service Departments or local distributors (see back cover or visit www.qiagen.com ).
Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of
guanidine thiocyanate. Guanidine salts can form highly reactive compounds when
combined with bleach. If liquid containing these buffers is spilt, clean with suitable
laboratory detergent and water. If the spilt liquid contains potentially infectious agents,
clean the affected area first with laboratory detergent and water, and then with 1% (v/v)
sodium hypochlorite.
Automated purification
Purification of RNA can be fully automated on the QIAcube®. The innovative QIAcube
uses advanced technology to process QIAGEN spin columns, enabling seamless
integration of automated, low-throughput sample prep into your laboratory workflow.
Sample preparation using the QIAcube follows the same steps as the manual procedure
(i.e., lyse, bind, wash, and elute) enabling you to continue using the RNeasy Micro Kit
for purification of high-quality RNA. For more information about the automated
procedure, see the relevant protocol sheet available at www.qiagen.com/MyQIAcube .
* For purification of miRNA and total RNA from a wide range of cells and tissues, we recommend using
miRNeasy Kits. For details, visit www.qiagen.com/miRNA .
R
RNeasy Micro
Procedure
Cells
Tissue LMD samples
Lyse and
➊ and ➋ homogenize
➌ Add ethanol
➎ Wash
➏ Elute
Concentrated
RNA solution
* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.
†
For ordering information, see page 61.
‡
If using proteinase K from another supplier, use a 20 mg/ml solution in water.
* Amounts can vary due to factors such as species, developmental stage, and growth conditions. Since the
RNeasy Micro procedure enriches for mRNA and other RNA species >200 nucleotides, the total RNA yield
does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15–20% of total
cellular RNA.
†
Using the protocol for purification of total RNA from fibrous tissues (page 30).
Carrier RNA
The RNeasy Micro Kit contains poly-A RNA for use as carrier RNA. When added to
lysates from very small samples, the carrier RNA may in some cases improve the
recovery of total RNA. Carrier RNA is not required when processing more than
500 cells or more than about 2 µg tissue.
As demonstrated in many different RT-PCR systems, the small amounts of poly-A RNA
used as carrier RNA in total RNA purification do not interfere with subsequent RT-PCR,
even when oligo-dT is used as a primer for reverse transcription. Reverse-transcription
reactions typically contain an excess of oligo-dT primers, and the small amounts of
poly-A used as carrier RNA are insignificant in comparison.
* RNA purified using poly-A RNA as carrier RNA is not compatible with Affymetrix kits for 3' in vitro
transcription, such as One-Cycle Target Labeling and Control Reagents; Two-Cycle Target Labeling and
Control Reagents; and GeneChip® HT One-Cycle Target Labeling and Controls Kit.
†
This is not a complete list of suppliers and does not include many important vendors of biological supplies.
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
Table 4. Growth area and number of HeLa cells in various culture vessels
* Per well, if multiwell plates are used; varies slightly depending on the supplier.
†
Cell numbers are given for HeLa cells (approximate length = 15 µm), assuming confluent growth. Numbers
will vary for different kinds of animal and human cells, which vary in length from 10 to 30 µm.
‡
This number of cells exceeds the binding capacity of the RNeasy MinElute spin columns. To process this
many cells, split the lysate into appropriate aliquots (≤5 x 105 cells each) and load them onto separate
RNeasy MinElute spin columns.
Cells
Important points before starting
I If using the RNeasy Micro Kit for the first time, read “Important Notes” (page 10).
I If preparing RNA for the first time, read Appendix A (page 50).
I If using the TissueRuptor, ensure that you are familiar with operating it by referring
to the TissueRuptor User Manual and TissueRuptor Handbook.
I Cell pellets can be stored at –70°C for later use or used directly in the procedure.
Determine the number of cells before freezing. Frozen cell pellets should be
thawed slightly so that they can be dislodged by flicking the tube in step 2.
Homogenized cell lysates from step 3 can be stored at –70°C for several months.
Frozen lysates should be incubated at 37°C in a water bath until completely
thawed and salts are dissolved. Avoid prolonged incubation, which may
compromise RNA integrity. If any insoluble material is visible, centrifuge for 5 min
at 3000–5000 x g. Transfer the supernatant to a new RNase-free glass or
polypropylene tube, and continue with step 4.
I Cells stored in RNAprotect Cell Reagent can also be used in the procedure.
Transfer the entire sample, including any material deposited at the bottom of the
storage vessel, to a centrifuge tube. Pellet the cells by centrifuging for 5 min at
5000 x g, and remove the supernatant by pipetting (if necessary, thaw the sample
before centrifuging). Proceed immediately to step 2.
I Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not
compatible with disinfecting reagents containing bleach. See page 6 for safety
information.
I Perform all steps of the procedure at room temperature (15–25°C). During the
procedure, work quickly.
I Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure
that the centrifuge does not cool below 20°C.
Cells
centrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefully
remove all supernatant by aspiration, and proceed to step 2.
Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the
lysate, affecting the conditions for binding of RNA to the RNeasy MinElute
membrane. Both effects may reduce RNA yield.
1b. Cells grown in a monolayer (do not use more than 5 x 105 cells):
Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or
trypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture
flasks should always be trypsinized.
To lyse cells directly:
Determine the number of cells. Completely aspirate the cell-culture medium, and
proceed immediately to step 2.
Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the
lysate, affecting the conditions for binding of RNA to the RNeasy MinElute
membrane. Both effects may reduce RNA yield.
To trypsinize and collect cells:
Determine the number of cells. Aspirate the medium, and wash the cells with PBS.
Aspirate the PBS, and add 0.1–0.25% trypsin in PBS. After the cells detach from
the dish or flask, add medium (containing serum to inactivate the trypsin), transfer
the cells to an RNase-free glass or polypropylene centrifuge tube (not supplied),
and centrifuge at 300 x g for 5 min. Completely aspirate the supernatant, and
proceed to step 2.
Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the
lysate, affecting the conditions for binding of RNA to the RNeasy MinElute
membrane. Both effects may reduce RNA yield.
2. Disrupt the cells by adding Buffer RLT.
For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350 µl
Buffer RLT (if processing ≤1 x 105 cells, add 75 µl Buffer RLT instead). Vortex or
pipet to mix, and proceed to step 3.
Note: Incomplete loosening of the cell pellet may lead to inefficient lysis and
reduced RNA yields.
to step 3.
3. Homogenize the lysate according to step 3a, 3b, or 3c.
See “Disrupting and homogenizing starting material”, page 12, for more details
on homogenization. If processing ≤1 x 105 cells, homogenize by vortexing for
1 min. After homogenization, proceed to step 4.
Note: If processing <500 cells, 20 ng carrier RNA (5 µl of a 4 ng/µl solution) may
be added to the lysate before homogenization. Prepare the carrier RNA as
described in “Things to do before starting”.
Note: Incomplete homogenization leads to significantly reduced RNA yields and
can cause clogging of the RNeasy MinElute spin column. Homogenization with
the TissueRuptor or QIAshredder homogenizer generally results in higher RNA
yields than with a syringe and needle.
3a. Pipet the lysate directly into a QIAshredder spin column (not supplied) placed in a
2 ml collection tube, and centrifuge for 2 min at full speed. Proceed to step 4.
3b. Place the tip of the TissueRuptor disposable probe into the lysate and operate the
TissueRuptor at full speed until the lysate is homogenous (usually 30 s). Proceed to
step 4.
Note: To avoid damage to the TissueRuptor and disposable probe during
operation, make sure the tip of the probe remains submerged in the buffer.
3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter)
fitted to an RNase-free syringe. Proceed to step 4.
4. Add 1 volume of 70% ethanol to the lysate, and mix well by pipetting. Do not
centrifuge. Proceed immediately to step 5.
Note: The volume of lysate may be less than 350 µl due to loss during
homogenization. If only 75 µl of Buffer RLT was used in step 2, then add only 75 µl
of 70% ethanol in this step.
Note: When purifying RNA from certain cell lines, precipitates may be visible after
addition of ethanol. This does not affect the procedure.
5. Transfer the sample, including any precipitate that may have formed, to an RNeasy
MinElute spin column placed in a 2 ml collection tube (supplied). Close the lid
gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-
through.*
* Flow-through contains Buffer RLT and is therefore not compatible with bleach. See page 6 for safety
information.
Cells
and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.*
Reuse the collection tube in step 9.
Optional: If on-column DNase digestion is not desired, add 700 µl Buffer RW1
instead, centrifuge for 15 s at ≥8000 x g, and discard the flow-through and
collection tube.* Proceed to step 10.
7. Add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the
tube.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only
be carried out by gently inverting the tube. Do not vortex.
8. Add the DNase I incubation mix (80 µl) directly to the RNeasy MinElute spin column
membrane, and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy MinElute
spin column membrane. DNase digestion will be incomplete if part of the mix sticks
to the walls or the O-ring of the spin column.
9. Add 350 µl Buffer RW1 to the RNeasy MinElute spin column. Close the lid gently,
and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through and collection tube.*
10. Place the RNeasy MinElute spin column in a new 2 ml collection tube (supplied).
Add 500 µl Buffer RPE to the spin column. Close the lid gently, and centrifuge for
15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard
the flow-through.
Reuse the collection tube in step 11.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to
Buffer RPE before use (see “Things to do before starting”).
11. Add 500 µl of 80% ethanol to the RNeasy MinElute spin column. Close the lid
gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin
column membrane. Discard the flow-through and collection tube.
Prepare the 80% ethanol with ethanol (96–100%) and the RNase-free water
supplied with the kit.
* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety
information.
Tissues
RNA yield and purity. A maximum amount of 5 mg fresh or frozen tissue or 2–3 mg
RNAlater or Allprotect stabilized tissue (which is partially dehydrated) can generally be
processed. For most tissues, the RNA binding capacity of the RNeasy MinElute spin
column and the lysing capacity of Buffer RLT will not be exceeded by these amounts.
Typical RNA yields from various tissues are given in Table 2 (page 11).
Some tissues such as spleen, parts of brain, lung, and thymus tend to form precipitates
during the procedure. However, this does not affect RNA purification.
Do not overload the RNeasy MinElute spin column, as this will significantly reduce RNA
yield and quality.
Weighing tissue is the most accurate way to quantitate the amount of starting material.
As a guide, a 1.5 mm cube (3.4 mm3) of most animal tissues weighs 3.5–4.5 mg.
I Perform all steps of the procedure at room temperature (15–25°C). During the
procedure, work quickly.
I Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure
that the centrifuge does not cool below 20°C.
Tissues
9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not
refreeze the aliquots after thawing.
Procedure
1. Excise the tissue sample from the animal or remove it from storage. Determine the
amount of tissue. Do not use more than 5 mg. Proceed immediately to step 2.
Weighing tissue is the most accurate way to determine the amount. If necessary,
cut the tissue on a clean surface and weigh the piece to be used.
For RNAlater or Allprotect stabilized tissues: Remove the tissue from the
stabilization reagent using forceps and be sure to remove any crystals that may
have formed. RNA in RNAlater or Allprotect stabilized tissues is protected during
cutting and weighing of tissues at room temperature (15–25°C). It is not necessary
to cut the tissues on ice or dry ice or in a refrigerated room. Remaining tissues can
be stored in RNAlater or Allprotect Reagent. Previously stabilized tissues can be
stored at –80°C without the reagent.
For unstabilized fresh or frozen tissues: RNA in harvested tissues is not protected
until the tissues are treated with RNAlater or Allprotect Reagent, flash-frozen, or
disrupted and homogenized in step 2. Frozen tissues should not be allowed to
thaw during handling. The relevant procedures should be carried out as quickly
as possible. Remaining fresh tissues can be placed into RNAlater Reagent to
stabilize RNA or in Allprotect Tissue Reagent to stabilize DNA, RNA, and protein.
However, previously frozen tissues thaw too slowly in the reagent, preventing the
reagent from diffusing into the tissues quickly enough to prevent RNA degradation.
2. Disrupt the tissue and homogenize the lysate in Buffer RLT (do not use more than
5 mg tissue) according to step 2a, 2b, or 2c.
See “Disrupting and homogenizing starting material”, page 12, for more details
on disruption and homogenization.
Note: Ensure that β-ME (or DTT) is added to Buffer RLT before use (see “Things to
do before starting”).
other methods.
2a. Disruption and homogenization using the TissueRuptor:
I Place the tissue in a suitably sized vessel. Add 350 µl Buffer RLT.
Note: Use a suitably sized vessel with sufficient extra headspace to
accommodate foaming, which may occur during homogenization.
Generally, round-bottomed tubes allow more efficient disruption and
homogenization than conical-bottomed tubes.
I Place the tip of the disposable probe into the vessel and operate the
TissueRuptor at full speed until the lysate is homogeneous (usually 30 s).
Proceed to step 3.
Note: To avoid damage to the TissueRuptor and disposable probe during
operation, make sure the tip of the probe remains submerged in the buffer.
Foaming may occur during homogenization. If this happens, let the
homogenate stand at room temperature for 2–3 min until the foam subsides
before continuing with the procedure.
2b. Disruption and homogenization using the TissueLyser:
I Place the tissues in 2 ml microcentrifuge tubes containing one stainless steel
bead (5 mm mean diameter).
If handling fresh or frozen tissue samples, keep the tubes on dry ice.
I Place the tubes at room temperature. Immediately add 350 µl Buffer RLT per
tube.
I Place the tubes in the TissueLyser Adapter Set 2 x 24.
I Operate the TissueLyser for 2 min at 20 Hz.
The time depends on the tissue being processed and can be extended until
the tissue is completely homogenized.
I Rearrange the collection tubes so that the outermost tubes are innermost and
the innermost tubes are outermost. Operate the TissueLyser for another 2 min
at 20 Hz.
Rearranging the tubes allows even homogenization.
Tissues
I Add 350 µl Buffer RLT.
I Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml
collection tube, and centrifuge for 2 min at full speed. Alternatively, pass the
lysate at least 5 times through a blunt 20-gauge needle fitted to an RNase-
free syringe. Proceed to step 3.
3. Centrifuge the lysate for 3 min at full speed. Carefully transfer the supernatant to
a new microcentrifuge tube (not supplied) by pipetting. Use only this supernatant
(lysate) in subsequent steps.
In some preparations, very small amounts of insoluble material will be present after
the 3-min centrifugation, making the pellet invisible.
4. Add 1 volume (usually 350 µl) of 70% ethanol to the lysate, and mix well by
pipetting. Do not centrifuge. Proceed immediately to step 5.
Note: The volume of 70% ethanol to add may be less than 350 µl if some lysate
was lost during homogenization.
Note: Precipitates may be visible after addition of ethanol, but this does not affect
the procedure.
5. Transfer the sample, including any precipitate that may have formed, to an RNeasy
MinElute spin column placed in a 2 ml collection tube (supplied). Close the lid
gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-
through.*
Optional: If recovery of protein is desired, keep the flow-through on ice and follow
steps E1–E5 in Appendix E on page 60.
Reuse the collection tube in step 6.
* Flow-through contains Buffer RLT and is therefore not compatible with bleach. See page 6 for safety
information.
* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety
information.
Tissues
no ethanol is carried over during RNA elution.
13. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (supplied).
Add 14 µl RNase-free water directly to the center of the spin column membrane.
Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA.
As little as 10 µl RNase-free water can be used for elution if a higher RNA
concentration is required, but the yield will be reduced by approximately 20%.
Do not elute with less than 10 µl RNase-free water, as the spin column membrane
will not be sufficiently hydrated.
The dead volume of the RNeasy MinElute spin column is 2 µl: elution with 14 µl
RNase-free water results in a 12 µl eluate.
For RT-PCR and real-time RT-PCR with the purified RNA, QIAGEN offers a range
of optimized, ready-to-use kits that provide highly specific and sensitive results. For
details, visit www.qiagen.com/PCR . For whole transcriptome amplification (WTA)
of limited amounts of RNA, we recommend the QuantiTect Whole Transcriptome
Kit. For details, visit www.qiagen.com/goto/WTA .
permit proteinase K digestion, this protocol should not be used for tissues rich in RNases,
such as spleen or intestine. In general, the protocol on page 23 is the protocol of choice
for other tissues.
Fibrous Tissues
Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not
compatible with disinfecting reagents containing bleach. See page 6 for safety
information.
I Unless otherwise indicated, perform all steps of the procedure at room temperature
(15–25°C). During the procedure, work quickly.
I Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure
that the centrifuge does not cool below 20°C.
glass vial, divide it into single-use aliquots, and store at –30 to –15° for up to
9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not
refreeze the aliquots after thawing.
Procedure
1. Heat a water bath or heating block to 55°C for proteinase K digestion in step 5.
2. Excise the tissue sample from the animal or remove it from storage. Determine the
amount of tissue. Do not use more than 5 mg. Proceed immediately to step 3.
Weighing tissue is the most accurate way to determine the amount. If necessary,
cut the tissue on a clean surface and weigh the piece to be used.
For RNAlater or Allprotect stabilized tissues: Remove the tissue from the
stabilization reagent using forceps and be sure to remove any crystals that may
have formed. RNA in RNAlater or Allprotect stabilized tissues is protected during
cutting and weighing of tissues at room temperature (15–25°C). It is not necessary
to cut the tissues on ice or dry ice or in a refrigerated room. Remaining tissues can
be stored in RNAlater or Allprotect Reagent. Previously stabilized tissues can be
stored at –80°C without the reagent.
For unstabilized fresh or frozen tissues: RNA in harvested tissues is not protected
until the tissues are treated with RNAlater or Allprotect Reagent, flash-frozen, or
disrupted and homogenized in step 3. Frozen tissues should not be allowed to
thaw during handling. The relevant procedures should be carried out as quickly
as possible. Remaining fresh tissues can be placed into RNAlater Reagent to
stabilize RNA or in Allprotect Tissue Reagent to stabilize DNA, RNA, and protein.
However, previously frozen tissues thaw too slowly in the reagent, preventing the
reagent from diffusing into the tissues quickly enough to prevent RNA degradation.
Fibrous Tissues
can cause clogging of the RNeasy MinElute spin column. Homogenization with
the TissueRuptor or TissueLyser generally results in higher RNA yields than with
other methods.
3a. Disruption and homogenization using the TissueRuptor:
I Place the tissue in a suitably sized vessel. Add 150 µl Buffer RLT.
Note: Use a suitably sized vessel with sufficient extra headspace to
accommodate foaming, which may occur during homogenization.
Generally, round-bottomed tubes allow more efficient disruption and
homogenization than conical-bottomed tubes.
I Place the tip of the disposable probe into the vessel and operate the
TissueRuptor at full speed until the lysate is homogeneous (usually 30 s).
Proceed to step 4.
Note: To avoid damage to the TissueRuptor and disposable probe during
operation, make sure the tip of the probe remains submerged in the buffer.
Foaming may occur during homogenization. If this happens, let the
homogenate stand at room temperature for 2–3 min until the foam subsides
before continuing with the procedure.
3b. Disruption and homogenization using the TissueLyser:
I Place the tissues in 2 ml microcentrifuge tubes containing one stainless steel
bead (5 mm mean diameter).
If handling fresh or frozen tissue samples, keep the tubes on dry ice.
I Place the tubes at room temperature. Immediately add 150 µl Buffer RLT per
tube.
I Place the tubes in the TissueLyser Adapter Set 2 x 24.
I Operate the TissueLyser for 2 min at 20 Hz.
Fibrous Tissues
collection tube.* Proceed to step 14.
11. Add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the
tube.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only
be carried out by gently inverting the tube. Do not vortex.
12. Add the DNase I incubation mix (80 µl) directly to the RNeasy MinElute spin column
membrane, and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy MinElute
spin column membrane. DNase digestion will be incomplete if part of the mix sticks
to the walls or the O-ring of the spin column.
13. Add 350 µl Buffer RW1 to the RNeasy MinElute spin column. Close the lid gently,
and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through and collection tube.*
14. Place the RNeasy MinElute spin column in a new 2 ml collection tube (supplied).
Add 500 µl Buffer RPE to the spin column. Close the lid gently, and centrifuge for
15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard
the flow-through.
Reuse the collection tube in step 15.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to
Buffer RPE before use (see “Things to do before starting”).
15. Add 500 µl of 80% ethanol to the RNeasy MinElute spin column. Close the lid
gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin
column membrane. Discard the flow-through and collection tube.
* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6
for safety information.
interfere with downstream reactions. Centrifugation with the lids open ensures that
no ethanol is carried over during RNA elution.
17. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube (supplied).
Add 14 µl RNase-free water directly to the center of the spin column membrane.
Close the lid gently, and centrifuge for 1 min at full speed to elute the RNA.
As little as 10 µl RNase-free water can be used for elution if a higher RNA
concentration is required, but the yield will be reduced by approximately 20%.
Do not elute with less than 10 µl RNase-free water, as the spin column membrane
will not be sufficiently hydrated.
The dead volume of the RNeasy MinElute spin column is 2 µl: elution with 14 µl
RNase-free water results in a 12 µl eluate.
For RT-PCR and real-time RT-PCR with the purified RNA, QIAGEN offers a range
of optimized, ready-to-use kits that provide highly specific and sensitive results. For
details, visit www.qiagen.com/PCR . For whole transcriptome amplification (WTA)
of limited amounts of RNA, we recommend the QuantiTect Whole Transcriptome
Kit. For details, visit www.qiagen.com/goto/WTA .
Microdissected
I
Cryosections
To minimize RNA degradation, avoid prolonged storage of unstabilized samples
at room temperature. RNA in tissues is not protected before flash-freezing in liquid
nitrogen.
I Tissue lysates from step 4 can be stored at –70°C for several months. Incubate
frozen lysates at 37°C in a water bath until completely thawed and salts are
dissolved before continuing with step 5. Avoid prolonged incubation, which may
compromise RNA integrity.
I Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not
compatible with disinfecting reagents containing bleach. See page 6 for safety
information.
I Perform all steps of the procedure at room temperature (15–25°C). During the
procedure, work quickly.
I Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure
that the centrifuge does not cool below 20°C.
I In the procedure below, L refers to use of the Leica® AS LMD System (which
requires reduced buffer volumes), and G refers to use of other laser microdissection
systems.
Microdissected
Cryosections
homogenization.
Note: Precipitates may be visible after addition of ethanol. This does not affect the
procedure.
6. Transfer the sample, including any precipitate that may have formed, to an RNeasy
MinElute spin column placed in a 2 ml collection tube (supplied). Close the lid
gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-
through.*
Reuse the collection tube in step 7.
7. Add 350 µl Buffer RW1 to the RNeasy MinElute spin column. Close the lid gently,
and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.*
Reuse the collection tube in step 10.
Optional: If on-column DNase digestion is not desired, add 700 µl Buffer RW1
instead, centrifuge for 15 s at ≥8000 x g, and discard the flow-through and
collection tube.* Proceed to step 11.
* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6
for safety information.
* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety
information.
Microdissected
Cryosections
information.
I Perform all steps of the procedure at room temperature (15–25°C). During the
procedure, work quickly.
I Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure
that the centrifuge does not cool below 20°C.
I In the procedure below, L refers to use of starting volumes ≤100 µl, and G refers
to use of starting volumes of 100–200 µl.
Procedure
1. Adjust the sample to a volume of L 100 µl or G 200 µl with RNase-free water.
Add L 350 µl or G 700 µl Buffer RLT, and mix well.
If starting with an RNA pellet, be sure that the pellet is dissolved in the RNase-free
water (supplied) before adding Buffer RLT.
Optional: Add β-ME (or DTT) to Buffer RLT before use (see “Things to do before
starting”).
2. Add L 250 µl or G 500 µl of 96–100% ethanol to the diluted RNA, and mix well
by pipetting. Do not centrifuge. Proceed immediately to step 3.
3. Transfer the sample (700 µl) to an RNeasy MinElute spin column placed in a 2 ml
collection tube (supplied). Close the lid gently, and centrifuge for 15 s at
≥8000 x g (≥10,000 rpm). Discard the flow-through.*
For G samples >700 µl, transfer the remaining sample (up to 700 µl) and repeat
the centrifugation. Discard the flow-through.*
RNA Cleanup
4. Optional: Add 700 µl Buffer RW1 to the RNeasy MinElute spin column. Close the
lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin
column membrane. Discard the flow-through and collection tube.*
5. Place the RNeasy MinElute spin column in a new 2 ml collection tube (supplied).
Add 500 µl Buffer RPE to the spin column. Close the lid gently, and centrifuge for
15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard
the flow-through.
Reuse the collection tube in step 6.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to
Buffer RPE before use (see “Things to do before starting”).
6. Add 500 µl of 80% ethanol to the RNeasy MinElute spin column. Close the lid
gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin
column membrane. Discard the flow-through and collection tube.
* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6
for safety information.
General handling
Proper microbiological, aseptic technique should always be used when working with
RNA. Hands and dust particles may carry bacteria and molds and are the most common
sources of RNase contamination. Always wear latex or vinyl gloves while handling
reagents and RNA samples to prevent RNase contamination from the surface of the skin
or from dusty laboratory equipment. Change gloves frequently and keep tubes closed
whenever possible. Keep purified RNA on ice when aliquots are pipetted for
downstream applications.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the
procedure. These tubes are generally RNase-free and do not require pretreatment to
inactivate RNases.
Nondisposable plasticware
Nondisposable plasticware should be treated before use to ensure that it is RNase-free.
Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA* followed
by RNase-free water (see ”Solutions”, page 51). Alternatively, chloroform-resistant
plasticware can be rinsed with chloroform* to inactivate RNases.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
Electrophoresis tanks
Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS),*
thoroughly rinsed with RNase-free water, and then rinsed with ethanol† and allowed to
dry.
Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a
strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of
0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and
water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml
of the solution to be treated and shake vigorously to bring the DEPC into solution. Let
the solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove any
trace of DEPC. DEPC will react with primary amines and cannot be used directly to treat
Tris buffers. DEPC is highly unstable in the presence of Tris buffers and decomposes
rapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first,
and then dissolve Tris* to make the appropriate buffer. Trace amounts of DEPC will
modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated
with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or
RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues
have been modified. Residual DEPC must always be eliminated from solutions or vessels
by autoclaving or heating to 100°C for 15 minutes.
Note: RNeasy buffers are guaranteed RNase-free without using DEPC treatment and are
therefore free of any DEPC contamination.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
†
Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the
supplier’s instructions.
Quantification of RNA
The concentration of RNA can be determined by measuring the absorbance at 260 nm
(A260) in a spectrophotometer (see “Spectrophotometric quantification of RNA” below).
For small amounts of RNA, however, it may not be possible to accurately determine
amounts photometrically. Small amounts of RNA can be quantified using an Agilent®
2100 bioanalyzer, fluorometric quantification, or quantitative, real-time RT-PCR. When
purifying RNA from particularly small samples (e.g., laser-microdissected samples),
quantitative, real-time RT-PCR should be used for quantification.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
Purity of RNA
The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of
the purity of RNA with respect to contaminants that absorb in the UV spectrum, such as
protein. However, the A260/A280 ratio is influenced considerably by pH. Since water is
not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results
in a lower A260/A280 ratio and reduced sensitivity to protein contamination.* For
accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure
RNA has an A260/A280 ratio of 1.9–2.1† in 10 mM Tris·Cl, pH 7.5. Always be sure to
calibrate the spectrophotometer with the same solution used for dilution.
For determination of RNA concentration, however, we recommend dilution of the
sample in a buffer with neutral pH since the relationship between absorbance and
concentration (A260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient
calculated for RNA at neutral pH (see “Spectrophotometric quantification of RNA”,
page 52).
DNA contamination
No currently available purification method can guarantee that RNA is completely free
of DNA, even when it is not visible on an agarose gel. While the RNeasy Micro Kit will
remove the vast majority of cellular DNA, trace amounts may still remain in the purified
RNA, depending on the amount and nature of the sample.
For analysis of very low abundance targets, any interference by residual DNA
contamination can be detected by performing real-time RT-PCR control experiments in
which no reverse transcriptase is added prior to the PCR step.
* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the
spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.
†
Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some
spectrophotometers.
Integrity of RNA
The integrity and size distribution of total RNA purified with RNeasy Kits can be checked
by denaturing agarose gel electrophoresis and ethidium bromide staining* or by using
an Agilent 2100 bioanalyzer. The respective ribosomal RNAs should appear as sharp
bands or peaks. The apparent ratio of 28S rRNA to 18S RNA should be approximately
2:1. If the ribosomal bands or peaks of a specific sample are not sharp, but appear as
a smear towards smaller sized RNAs, it is likely that the RNA sample suffered major
degradation either before or during RNA purification.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
* This protocol also works well with some other reagents containing phenol and guanidine thiocyanate.
Please contact QIAGEN Technical Services for more details (see back cover for contact information).
Procedure
C1. Carry out homogenization of the sample in QIAzol Lysis Reagent, followed by
phase separation, as described in the QIAzol Handbook (steps 1–7 of the QIAzol
protocol for lysis and homogenization).
C2. Transfer the upper, aqueous phase to a new collection tube. Add 1 volume of 70%
ethanol, and mix thoroughly by vortexing. Do not centrifuge. Proceed
immediately to step C3.
Note: If processing <2 µg tissue, 20 ng carrier RNA (5 µl of a 4 ng/µl solution)
may be added to the aqueous phase before adding ethanol. Prepare the carrier
RNA as described in “Things to do before starting”.
* Flow-through contains QIAzol Lysis Reagent or Buffer RW1 and is therefore not compatible with bleach.
See the QIAzol Handbook and page 6 for safety information.
Procedure
D1. Mix the following in a microcentrifuge tube:
I ≤87.5 µl RNA solution (contaminated with genomic DNA)
I 10 µl Buffer RDD
I 2.5 µl DNase I stock solution
Make the volume up to 100 µl with RNase-free water.
The reaction volumes can be doubled if necessary (to 200 µl final volume).
D2. Incubate on the benchtop (20–25°C) for 10 min.
D3. Clean up the RNA according to “Protocol: RNA Cleanup and Concentration” on
page 42.
Procedure
Bind total RNA to the RNeasy MinElute spin column as described in the cell protocol
(from page 16, steps 1–5), the tissue protocol (from page 23, steps 1–5), or the fibrous
tissue protocol (from page 30, steps 1–9). Then follow steps E1–E5 below to precipitate
protein from the flow-through.
E1. Add 4 volumes of ice-cold acetone to the flow-through from the RNeasy MinElute
spin column.
E2. Incubate for 30 min on ice or at –30 to –15°.
E3. Centrifuge for 10 min at full speed in a benchtop centrifuge. Discard the
supernatant and air-dry the pellet.†
E4. Optional: Wash the pellet with 100 µl ice-cold ethanol and air-dry.
Do not overdry the pellet as this may make resuspension more difficult.
E5. Resuspend the pellet in the buffer for your downstream application.
Sodium dodecyl sulfate (SDS) causes guanidine salts to precipitate. In case the
pellet contains traces of guanidine thiocyanate, load the sample onto an SDS-
PAGE gel immediately after heating for 7 minutes at 95°C.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
†
Supernatant contains guanidine thiocyanate and is therefore not compatible with bleach. See page 6 for
safety information.
* Visit www.qiagen.com/automation to find out more about the TissueRuptor and TissueLyser and to order.
For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services
or your local distributor.
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